Although RhoA and RhoC have been implicated in invasion and metastasis, the molecular mechanisms behind Rho-mediated metastasis are unclear, and it is unknown which metastatic processes involve Rho proteins. Our long-term goals for this project are to understand the importance of these two proteins during key stages of the metastatic cascade, and to identify which Rho effector pathways are involved. We hypothesize that both RhoA and RhoC will promote the spread of breast cancer by enhancing multiple stages of metastasis. We further predict that Rho-mediated metastasis will require specific Rho effectors, and that different Rho effectors may mediate each Rho-mediated process during metastasis. Specific Aims: (1) Determine if breast cancer metastasis requires RhoA or RhoC, and identify which stages of the metastatic cascade are dependent upon these proteins. (2) Identify which stages of breast cancer metastasis are promoted by increased expression of RhoA or RhoC. (3) Identify Rho effectors that are required for Rho-mediated metastasis. (4) Test the involvement of RhoA, RhoC, and Rho effectors during mammary carcinoma development, progression, and metastasis. Study design: We will use retroviral expression constructs and miR30-based shRNAs to alter the expression of RhoA or RhoC in a series of mammary carcinoma (MC) cell lines with varying metastatic potentials. Cells will then be implanted into the mammary fat pads of syngeneic mice to assay differences in several metastatic processes (invasion, intravasation, extravasation and metastatic tumor formation). We will also screen for Rho effectors involved in metastasis by orthotopically implanting pools of MC cells expressing barcoded miR30-based shRNAs that each target a known Rho effector, and then screening for enrichment or dramatic reduction of each barcode in metastatic lesions that form in the lungs. As an additional model system, we will also isolate primary MC cells from tumor-bearing MMTV Polyoma middle T mice, manipulate Rho protein and effector expression, and then orthotopically implant the cells into donor mice and assay the effects on metastatic processes. PUBLIC HEALTH RELEVANCE: Breast cancer metastasis kills 40,000 American women each year, and until we fully understand how specific proteins influence the metastatic processes, we will be unable to design therapies that combat this disease. Our studies aim to identify which metastatic processes involve Rho proteins, as well as determine which molecular pathways downstream of Rho proteins are required for each of these processes.